Method for ameliorating pain by modification of NMDA receptors through inhibition of Src

ABSTRACT

The present invention provides a method for ameliorating inflammatory and/or neuropathic pain in a subject by modifying the activity of N-methyl-D-aspartate (NMDA) receptors in cells of the subject by inhibition of the interaction of the unique domain of the tyrosine kinase Src enzyme and the NMDA receptor complex.

CROSS REFERENCE TO RELATED APPLICATION

This application is a continuation-in-part of application Ser. No.10/814,109, filed on Mar. 30, 2004, now U.S. Pat. No. 7,425,540, thecontents of which is herein incorporated by reference.

FIELD OF THE INVENTION

The instant invention relates generally to protein-protein interactionsthat regulate intra and intercellular communication; particularly tomethods for modification of protein-protein interactions; and mostparticularly to a method for ameliorating pain in a subject by modifyingthe activity of NMDA (N-methyl-D-aspartate) receptors located in cellsby inhibition of the interaction of the unique domain of the tyrosinekinase Src enzyme with proteins of the NMDAR complex.

BACKGROUND OF THE INVENTION

Excitatory transmission at central synapses is primarily mediated by theamino acid glutamate acting through postsynaptic ionotropic receptors(Dingledine et al. Pharmacological Review 51:7-61 1999). TheN-methyl-D-aspartate receptor (NMDAR) is one such type of ionotropicglutamate receptor (Dingledine et al. Pharmacological Review 51:7-611999). NMDARs are multiprotein complexes located at excitatory synapseswithin the postsynaptic density (PSD) comprised of the core channelsubunits together with associated scaffolding and regulatory proteinsthat control receptor localization, ionic flux through the receptor anddownstream signaling events (Scannevin et al. Nature ReviewsNeuroscience 1:133-141 2000; Sheng et al. Annual Review of Physiology62:755-778 2000). NMDAR's are crucial for central nervous system (CNS)development, neuroplasticity and pathophysiology (Dingledine et al.Pharmacological Review 51:7-61 1999; Sheng et al. Science 298:776-7802002). Multiple factors regulate NMDAR function, including dynamiccycling of protein phosphorylation and dephosphorylation atserine/theronine or tyrosine residues (Wang et al. Nature 369:233-2351994; Smart Current Opinion in Neurobiology 7:358-367 1997). The Srcprotein is one such factor that modulates the activity of the NMDARs (Yuet al. Science 275:674-678 1997; Lu et al. Science 279:1363-1368 1998;Yu et al. Nature 396:469-474 1998).

The non-receptor protein tyrosine kinase Src is a ubiquitous enzyme withkey roles in diverse development, physiological and pathologicalprocesses (Brown et al. Biochim. Biophys. Acta 1287:121-149 1996).Domains identified in Src- the Src homology 3 (SH3) domain, the SH2domain and the SH1 (catalytic) domain are signature regions that havebeen used to define highly-conserved protein modules found in a widevariety of signaling proteins (Pawson Nature 373:573-580 1995). Inaddition to these highly-conserved regions, Src also contains a regionof low sequence conservation and unknown function, termed the uniquedomain.

Src is highly expressed in the CNS, functioning to regulateglutamatergic neurotransmission and synaptic plasticity (Ali et al.Current Opinion in Neurobiology 11:336-342 2001; Salter and Kalia NatureReviews:Neuroscience 5:317-328 2004)). At glutamatergic synapses, Srcmodulates the activity of NMDARs (Yu et al. Science 275:674-678 1997; Luet al. Science 279:1363-1368 1998; Yu et al. Nature 396:469-474 1998).Src represents a point through which multiple signaling cascades fromG-protein coupled receptors (Luttrell et al. Journal of Cell Science115:455-465 2002), Eph receptors (Henderson et al. Neuron 32:1041-10562001; Takasu et al. Science 295:491-495 2002; Murai et al. Neuron33:159-162 2002) and integrins (Lin et al. Journal of Neurophysiology89:2874-2878 2003; Kramar et al. Journal of Biological Chemistry278:10722-10730 2003) converge to upregulate NMDAR channel activity,thus mediating essential neuronal excitation. The upregulation of NMDARactivity by Src is necessary for long-term potentiation (LTP) ofsynaptic transmission at Schaffer collateral-CA1 neuron synapses in thehippocampus (Ali et al. Current Opinion in Neurobiology 11:336-3422001), the predominant cellular model for learning and memory (KandelScience 294:1030-1038 2001).

However, abnormal regulation of NMDARs can have numerous pathologiceffects; most resulting from the production of nitric oxide, a signalingmolecule which mediates excitotoxicity (Dawson et al. Proceedings of theNational Academy of Science USA 88:6368 1991). NMDARS mediate ischemicbrain injury, as seen, for example in stroke and traumatic injury (Simonet al. Science 226:850 1984). In addition, abnormal NMDAR regulation hasbeen implicated in Alzheimer's disease, Parkinson's disease (Coyle etal. Science 262:689 1993), schizophrenia (Hirsch et al. PharmacologyBiochemistry and Behavior 56(4):797-802 1997), epilepsy (U.S. Pat. No.5,914,403), glaucoma (US Application. 2002 0077322 A1) and chronic pain(Guo et al. Journal of Neuroscience 22:6208-6217 2002).

Although NMDARs are implicated in numerous pathological conditions,non-selective blocking of their function is deleterious, since completeblockade of synaptic transmission mediated by NMDA receptors is known tohinder neuronal survival (Ikonomidou et al. Lancet: Neurology 1:383-3862002; Fix et al. Experimental Neurology 123:204 1993; Davis et al.Stroke 31:347 2000; Morris et al. Journal of Neurosurgery 91:737 1999).

Additionally, inhibition of Src kinases may also have deleteriousresults. Since kinases play a part in the regulation of cellularproliferation, they are frequently targeted for the development of newcancer therapies.

The majority of these therapies inhibit function of the kinase catalyticdomain, which is often highly conserved between distinct kinases. Thus,inhibition of Src in the CNS with a standard kinase inhibitor maycross-react with additional kinases and adversely affect normal neuronalfunctions.

Considering the above-mentioned deleterious effects resulting fromdirect blockage of NMDARs and/or indirect inhibition of NMDARs throughthe use of kinase inhibitors, it is clear that there remains a need inthe art for a method of modifying NMDARs which can attenuate downstreamNMDAR signaling, without completely blocking, ion-channel activity.

DESCRIPTION OF THE PRIOR ART

Since the NMDA receptor is critical to both normal neuronal function andpathology, there are many known methods for modification of NMDAreceptors; several examples of which are noted below.

U.S. Pat. No. 5,888,996 (David Farb) discloses a method for inhibitingNMDA glutamate receptor-mediated ion channel activity by treatment withan effective amount of a derivative of pregnenolone sulfate. This patentalso discloses a method for modulating/altering excitatoryglutamate-mediated synaptic activity by contacting neurons withpregnenolone sulfate or a derivative of pregnenolone sulfate.

U.S. Pat. No. 5,914,403 (Nichols et al.) discloses agents capable ofmodifying neuroexcitation through excitatory amino acid antagonists; inparticular quinolinic acid derivatives antagonistic to a glycine bindingsite in the NMDAR complex. The agents disclosed by Nichols et al. haveanticonvulsant activity.

U.S. Pat. No. 4,994,446 (Sokolovsky et al.) discloses a drug systemcomprising a MK-801/PCP type drug administered in combination with/or insequence with excitatory amino acids such as, glutamate, glycine,aspartate and analogs thereof. The excitatory amino acids facilitatebinding of the drug to the NMDAR channels. This drug system hasanticonvulsant activity and can alleviate brain damage due to stroke.

U.S. Pat. No. 6,653,354 (Franks et al.) discloses a method for reducingthe level of NMDAR activation by use of the NMDA antagonist, xenon toinhibit synaptic plasticity. The xenon composition of Franks et al. alsoacts as a neuroprotectant.

US Patent Application 2002 0123510 A1 (Chenard et al.) discloses amethod for treatment of traumatic brain injury (TBI) and stroke byadministration of a NR2B subtype selective NMDAR antagonist incombination with either of the following agents; sodium channelantagonist, nitric oxide synthase inhibitor, glycine site antagonist,potassium channel opener, AMPA/kainate receptor antagonist, calciumchannel antagonist, GABA-A receptor modulator, anti-inflammatory agentor a thrombolytic agent. These agents either protect neurons from toxicinsult, inhibit inflammatory responses after brain damage or promotecerebral reperfusion after hypoxia or ischemia.

Planells-Cases et al. (Mini Review of Medicinal Chemistry 3(7):749-7562003) disclose that small molecule antagonists of the NMDAR are usefulfor the treatment of neuropathic pain caused by injury to the peripheralor central nervous system.

US Patent Application 2002 0077322 A1 (George Ayoub) discloses methodsfor protecting neuronal cells from glutamate-induced toxicity, such asthat which occurs in ischemia and glaucoma, by increasing the activityof a cannabinoid agonist which binds specifically to a cannabinoidreceptor.

US Patent Application 2003 0050243 A1 (Michael Tymianski) discloses amethod for inhibition of binding between NMDARs and neuronal proteins.The inhibition is created by administration of a peptide replacement ofeither an NMDAR or neuronal protein interaction domain. Post-synapticdensity protein 95 (PSD-95) couples NMDARs to pathways mediatingexcitotoxicity and ischemic brain damage. The method of Tymianskiinvolves transducing neurons with peptides that bind modular domains oneither side of the NMDAR/PSD-95 interaction complex. This transductionattenuates downstream NMDAR signaling without blocking receptoractivity, protects cortical neurons from ischemic insult and reducescerebral infarction in rats exposed to transient focal cerebralischemia. This treatment was effective in the rats when applied beforeor one hour after the ischemic insult. (Aarts et al. Science 298:846-8502002) also discloses the research described in US Patent Application2003 0050243 A1.

As is exemplified by the examples listed above, the majority of knownmethods for modification of NMDA receptors generally involveadministration of receptor antagonists which inhibit receptor functioncompletely. The instant inventors are the first to modify the NMDAR byinhibiting the interaction of the unique domain of the tyrosine kinaseSrc enzyme with NADH dehydrogenase subunit 2 (ND2); thus preventing Srcupregulation of the NMDAR by preventing binding between Src and ND2.

SUMMARY OF THE INVENTION

Src-mediated upregulation of NMDAR activity is prevented by peptidefragments of the Src unique domain and by a unique domain-bindingantibody (Yu et al. Science 275:674-678 1997; Lu et al. Science279:1363-1368 1998) leading to the hypothesis that the upregulation ofNMDAR function by Src depends on an interaction between a region in theunique domain of Src and an unknown protein in the NMDAR complex (Ali etal. Current Opinion in Neurobiology 11:336-342 2001). In order to testthe hypothesis, the instant inventors searched for proteins that mayinteract with the unique domain of Src and may thereby mediate theinteraction between this kinase and NMDARs. These proteins weregenerally termed “SUDAPIs” (Src unique domain anchoring proteininhibitors) by the instant inventors since they anticipate that othersuch inhibitors may exist which exhibit identical functions.

As a result of their search, the instant inventors became the first toidentify NADH dehydrogenase subunit 2 (ND2; nucleotide sequence SEQ IDNO:8 and amino acid sequence SEQ ID NO:9) as a Src uniquedomain-interacting protein (Gingrich et al. PNAS 101(16):6237-62422004). ND2 functions as an adapter protein anchoring Src to the NMDARcomplex, thus permitting Src-mediated upregulation of NMDAR activity.The instant inventors identified a region of the Src unique domain whichinteracts with ND2; a region located approximately at amino acidpositions 40-49 of the Src protein (SEQ ID NO:1). The exogeneous peptideinhibits the ability of ND2 to anchor the Src protein to the NMDARcomplex. This peptide, approximately 10 amino acids in length, has beennamed “SUDAPI-1” by the instant inventors, since it is the first suchpeptide discovered which functions to inhibit the Src unique domainanchoring protein. Administration of this exogeneous peptide preventsND2 interaction with the Src unique domain; thus inhibiting Src-mediatedupregulation of NMDAR activity. Since this peptide alone cannot crossthe cell membrane to enter the cellular interior, it is combined with acarrier capable of penetrating the cell membrane. Illustrative, albeitnon-limiting examples of carriers are peptides derived from viraltransduction domains, such as the TAT domain derived from the HumanImmunodeficiency Virus (HIV) and VP22 derived from the Herpes SimplexVirus, arginine-rich peptides, fusogenic antennapedia peptides derivedfrom Drosophilia and lipids. Lipids can facilitate crossing of the cellmembrane by enclosing the peptide in a lipid vesicle or liposome (lipidtransfection protocol) or the peptide can be directly modified withlipid groups. Use of the HIV-Tat domain peptide as a carrier isexemplified in the Examples described herein. SUDAPI-1 fused to theHIV-Tat domain is designated “TSUDAPI-1” (SEQ ID NO:2). The NMDARactivity is evoked by glutamate and is additionally regulated by manydistinct pathways other than the Src pathway. Inhibition of Srcsuppresses but does not completely inhibit the NMDAR as is apparent fromthe electrophysiologic measurements of receptor activity shown in FIGS.5D-F. Thus, the instant invention provides methods and compositions formodifying NMDAR function without completely blocking the receptor oradversely affecting other neuronal proteins with the use of generalizedkinase inhibitors. These methods and compositions may be used toameliorate diseases and/or other conditions related to NMDAR signaling.Illustrative, albeit non-limiting examples of such diseases and/or otherconditions are stroke, hypoxia, ischemia, multiple sclerosis,Huntington's chorea, Parkinson's disease, Alzheimer's disease,hyperglycemia, diabetes, traumatic injury, epilepsy, grand mal seizures,spasticity, cerebral palsy, asthma, cardiac arrest, maculardegeneration, mental diseases, schizophrenia, AIDS dementia complex,other dementias, AIDS wasting syndrome, inflammation, pain, opioidaddiction, cocaine addiction, alcohol addiction, other conditionsassociated with substance abuse and anorexia. An example of suchamelioration is illustrated in Example 7 wherein pain behaviors arereduced in rats treated with the composition of the instant inventionprior to undergoing the formalin test. Furthermore, in addition toreducing inflammatory pain, the composition of the instant inventioninhibits Src-mediated NMDAR upregulation-dependent neuropathic pain inrats and mice having peripheral nerve injury (Example 9).

Src upregulation of the NMDAR is involved in the pathway of long-termpotentiation (LTP)(Huang et al. Neuron 29:485-496 2001; Lu et al.Science 279:1363-1367 1998). Since LTP is considered a model forlearning and memory, the compositions of the instant invention arecontemplated for use in methods which elucidate mechanisms of learningand memory and/or enhance learning and memory.

The NMDAR is expressed almost exclusively in neurons; however theinteraction between Src and ND2 was shown to occur in multiple anddiverse tissues (Example 8 and FIGS. 10A-B). Thus, the instant inventorshypothesize that the Src-ND2 interaction has functions other thanregulation of NMDAR's and contemplate that the compositions of theinstant invention can be used in methods for the general inhibition ofSrc in multiple cell types.

Accordingly, it is an objective of the instant invention to provide amethod for modifying NMDAR interaction with non-receptor tyrosine kinaseSrc in any cell which expresses the NMDAR by providing a compositionincluding at least one SUDAPI and administering the composition to thecell in an amount effective to achieve modification of the NMDARinteraction with non-receptor tyrosine kinase Src in the cell whereinsaid modification ameliorates a disease or condition related to NMDARsignaling. The methods and compositions of the instant invention areparticularly suited to use with cells of the nervous system but can alsobe used with any cell which expresses the NMDAR.

It is another objective of the instant invention to provide apharmaceutical composition for modifying NMDAR interaction withnon-receptor tyrosine kinase Src in cells comprising at least one SUDAPIcombined with a pharmacologically acceptable solution or carrier.

It is also an objective of the instant invention to provide a method formodifying NMDAR interaction with non-receptor tyrosine kinase Src in anycell which expresses the NMDAR by providing a composition includingSUDAPI-1 and administering the composition to the cell in an amounteffective to achieve modification of the NMDAR interaction withnon-receptor tyrosine kinase Src in the cell wherein said modificationameliorates a disease or condition related to NMDAR signaling.

It is another objective of the instant invention to provide apharmaceutical composition for modifying NMDAR interaction withnon-receptor tyrosine kinase Src in cells comprising SUDAPI-1 combinedwith a pharmacologically acceptable solution or carrier.

It is yet another objective of the instant invention to provide a methodfor modifying NMDAR interaction with non-receptor tyrosine kinase Src inany cell which expresses the NMDAR by providing a composition includingTSUDAPI-1 and administering the composition to the cell in an amounteffective to achieve modification of the NMDAR interaction withnon-receptor tyrosine kinase Src in the cell wherein said modificationameliorates a disease or condition related to NMDAR signaling.

It is still another objective of the instant invention to provide apharmaceutical composition for modifying NMDAR interaction withnon-receptor tyrosine kinase Src in cells comprising TSUDAPI-1 combinedwith a pharmacologically acceptable solution.

It is another objective of the instant invention to provide an isolatedpeptide (ND2.1; SEQ ID NO:7) which interacts with the Src unique domainto anchor Src to the NMDAR complex thus permitting Src-mediatedupregulation of NMDAR activity.

It is still another objective of the instant invention to provide amethod for inhibiting non-receptor tyrosine kinase Src in cellsexpressing non-receptor tyrosine kinase Src by providing a compositionincluding at least one SUDAPI and administering the composition to thecells in an amount effective to achieve inhibition of non-receptortyrosine kinase Src in the cells.

It is another objective of the instant invention to provide apharmaceutical composition for inhibiting non-receptor tyrosine kinaseSrc in cells comprising at least one SUDAPI combined with apharmacologically acceptable solution or carrier.

It is another objective of the instant invention to provide acomposition useful in methods for elucidating the mechanisms of learningand memory.

It is yet another objective of the instant invention to provide acomposition useful in methods for enhancing learning and memory.

It is another objective of the instant invention to provide acomposition useful in methods for treating inflammatory pain.

It is yet another objective of the instant invention to provide acomposition useful in methods for treating neuropathic pain.

Another objective of the instant invention is to provide a method forameliorating inflammatory and/or neuropathic pain in a subject bymodifying NMDAR interaction with non-receptor tyrosine kinase Src in anysubject having cells which express the NMDAR by providing a compositionincluding at least one SUDAPI and administering the composition to thesubject in an amount effective to achieve modification of the NMDARinteraction with non-receptor tyrosine kinase Src in the cells toameliorate pain in the subject.

It is yet another objective of the instant invention to provide a methodfor ameliorating inflammatory and/or neuropathic pain in a subject bymodifying NMDAR interaction with non-receptor tyrosine kinase Src incells of the subject by providing a composition including SUDAPI-1 (SEQID NO:1) or TSUDAPI-1 (SEQ ID NO:2) and administering the composition tothe subject in an amount effective to achieve modification of the MNDARinteraction with non-receptor tyrosine kinase Src in the cells toameliorate pain in the subject.

Other objectives and advantages of the instant invention will becomeapparent from the following description taken in conjunction with theaccompanying drawings wherein are set forth, by way of illustration andexample, certain embodiments of the instant invention. The drawingsconstitute a part of this specification and include exemplaryembodiments of the present invention and illustrate various objects andfeatures thereof.

ABBREVIATIONS AND DEFINITIONS

The following list defines terms, phrases and abbreviations usedthroughout the instant specification. Although the terms, phrases andabbreviations are listed in the singular tense the definitions areintended to encompass all grammatical forms.

As used herein, the term “subject” refers to any organism having cellswhich express or are capable of expressing NMDA receptors.

As used herein, the term “modification” refers to any action and/ortreatment which alters the function of a protein.

As used herein, the term “inhibition” refers to any action and/ortreatment which operates against the full activity of a protein thusreducing and/or completely suppressing protein function.

As used herein, the term “interaction” refers to an action wherein twosubstances in close physical proximity act upon each other.

As used herein, the term “anchor” means to stabilize or secure firmly inplace.

As used herein, the term “isolated peptide” refers to a peptide whichhas been “altered by the hand of man” and separated from the co-existingmaterials of its natural state. An isolated peptide has been changed orremoved from its original environment or both.

As used herein, the abbreviation “CNS” refers to the central nervoussystem, which includes the brain, cranial nerves and the spinal cord.

As used herein, the abbreviation “PNS” refers to the peripheral nervoussystem, which is the network of nerves that connect the CNS to organs,muscles, vessels and glands. Peripheral nerve injury often results inabnormal neuropathic pain, hyperalgesia and/or allodynia.

As used herein, the term “excitatory neurotransmission” refers thepassage of signals from one neuron to another via chemical substances orelectrical impulses.

As used herein, the abbreviation “NMDAR” refers to theN-methyl-D-aspartate receptor, an ionotropic cation-ion specific,ligand-gated (glutamate-gated) ion channel which is activated by NMDA orNMDA-like ligands (agonist activation) such as glutamate. The NMDAR is amulti-protein complex including the core channel subunits withassociated scaffolding and regulatory proteins, located in theexcitatory synapses in the post-synaptic density. Activation of thereceptor opens the channel to allow cations (Ca⁺², Na⁺ and K⁺) to crossthe cellular membrane. “Upregulation of NMDAR activity” refers to theenhanced opening of the receptor ion channels.

As used herein, the term “Src” refers to a protein exhibitingtyrosine-specific kinase activity. The Src protein is involved incontrolling diverse cellular functions, including regulation of NMDARactivity.

As used herein, the term “phosphorylation” refers to a reversiblecovalent modification wherein a phosphate group (non-protein) isattached/detached to a protein. The addition and removal of thephosphate group causes changes in the tertiary structure of the proteinthat alter its activity.

As used herein, the abbreviation “PSD” refers to the post-synapticdensity, a specialized portion of the neuronal cytoskeleton, locatednear the post-synaptic membrane. The PSD provides a support matrix forsignal transduction.

As used herein, the abbreviation “LTP” refers to long term potentiation,an activity-dependent persistent enhancement of synaptic transmissionwhich is considered a model of learning and memory. The biochemicalsignaling cascade which results in LTP involves the activation of Srcwhich in turn, activates NMDARs.

As used herein, the abbreviation “ND2” refers to NADH dehydrogenasesubunit 2, a subunit of mitochondrial Complex I. The instant inventorwas the first to recognize that ND2 is present in the PSD and acts as anadaptor protein for anchoring Src to the NMDAR complex.

As used herein, the abbreviation “SUDAPI” refers to any substance whichfunctions as a Src unique domain anchoring protein inhibitor.

As used herein, the abbreviation “SUDAPI-1” refers to the first Srcunique domain anchoring protein inhibitor discovered by the instantinventors. SUDAPI-1 is a peptide, generally 10 amino acid residues inlength corresponding approximately to amino acid positions 40-49 of theSrc unique domain (SEQ ID NO:1).

As used herein, the phrase “corresponding approximately to amino acidpositions 40-49 of the Src unique domain” refers to the slightdifference which is possible in amino acid position numbering of the Srcprotein due to species variations and conventions within the artregarding whether the first methionine counts as a residue or not.

As used herein, the abbreviation “TSUDAPI-1” refers to SUDAPI-1 which iscombined with the carrier peptide, HIV-Tat (SEQ ID NO:2).

As used herein, the term “carrier” refers to any substance which isattached to another substance which alone cannot traverse the cellmembrane to enter the cellular interior. The carrier substance functionsto carry this other substance through the cellular membrane into thecellular interior. Illustrative, albeit non-limiting examples includelipids and peptides having transducing and/or fusogenic ability.

As used herein, the term “HIV-Tat” refers to the transduction domain ofthe human immunodeficiency virus (HIV); the causative agent of AcquiredImmunodeficiency Syndrome (AIDS). HIV-Tat peptide is often used as acarrier to transport molecules into cells.

As used herein, the term “VP22” refers to a transduction domain of theherpes simplex virus. VP22 peptide is often used as a carrier totransport molecules into cells.

As used herein, the term “antennapedia” refers to peptides derived fromDrosophilia which have fusogenic ability. Antennapedia peptide is oftenused as a carrier to transport molecules into cells.

The phrase “pharmaceutically acceptable” is used herein as described inU.S. Pat. No. 6,703,489. “Pharmaceutically acceptable” means approved bya regulatory agency or listed in a generally approved pharmacopeia foruse in animals and humans. Solutions are usually preferred when acomposition is administered intravenously. Illustrative, albeitnon-limiting examples of pharmaceutically acceptable solutions includewater, oils, saline, aqueous dextrose and glycerol.

As used herein, the phrase “amount effective” refers to an amount of acomposition sufficient to elicit a change in activity of the NMDAR.

As used herein, the phrase “ameliorate a disease and/or condition”refers to an action which causes symptoms of a disease and/or conditionto improve or become better.

As used herein, the phrase “ameliorating pain” refers to an action whichcauses pain symptoms to improve and/or disappear.

As used herein, the abbreviation “SH” refers to a Src homology domain;regions that have been used to define highly-conserved protein modulesfound in a wide variety of signaling proteins (T. Pawson Nature373:573-580 1995).

As used herein, the phrase “unique domain” refers to a Src domain havinglow sequence conservation and unknown function.

As used herein, the abbreviation “ND4” refers to NADH degydrogenasesubunit 4, an oxidoreductase protein, a component of mitochondrialComplex I (J E Walker Quarterly Reviews of Biophysics 25(3):253-3241992; Sazanov et al. Biochemistry 39:7229-7235 2000; Sazanov et al.Journal of Molecular Biology 302:455-464 200).

As used herein, the abbreviation “NdufA9” refers to NADH-UbiquinoneOxidoreductase 1 alpha subcomplex 9, also a subunit of mitochondrialcomplex I (NCBI GeneID:4704).

As used herein, the abbreviation “Cyto 1” refers to cytochrome c oxidasesubunit 1, an inner mitochondrial membrane protein that is part ofComplex IV (Marusich et al. Biochim. Biophys. Acta 1362:145-159 1997).

As used herein, the abbreviation “mEPSCs” refers to miniature excitatorypost-synaptic currents, a type of excitatory neurotransmission.

The terms “SUDAPI-1” and “Src40-49” are used interchangeably herein (SEQID NO:1).

The terms “TSUDAPI-1”; “Src40-49-Tat”; “Src40-49-HIV-Tat”;“Tat-Src40-49” and “HIV-Tat-Src40-49” are used interchangeably herein(SEQ ID NO:2).

The terms “Src40-58” and “scrambled Src40-58” are used repeatedlythroughout and refer to peptides comprising amino acid residues 40-58 ofSEQ ID NO:4.

The term “Src49-58” is used repeatedly throughout and refers to apeptide comprising amino acid residues 49-58 of SEQ ID NO:4.

As used herein, the term “pain” refers to an unpleasant sensation. Painhas both physical and emotional components. Pain is mediated by nerveswhich carry the pain impulses to the brain where their interpretationcan be influenced by many factors.

As used herein, the term “inflammatory pain” refers to pain associatedwith inflammation. Inflammation is the nonspecific immune response of anorganism to infection, irritation and/or injury. Inflammation ischaracterized by redness, swelling, warmth and pain. The formalin testas performed on rats in Example 7 is a model for inflammatory pain.

As used herein, the term “neuropathic pain” refers to pain directlyassociated with the nervous system resulting from damage to the nervesor other changes in the nervous system. Neuropathic pain is associatedwith changes in sensory processing (for example, with temperature andtouch) and occurs without inflammation. The cuff-implantation in mice asexemplified in Example 9 is a model for neuropathic pain.

As used herein, the term “allodynia” refers to pain caused by a stimulusthat does not normally result in a pain response. Allodynia involves achange in the quality of a sensation or a loss of specificity of asense, i.e. a first response to a stimulus is not pain, but a nextresponse to the stimulus results in pain. Tactile allodynia is pain thatresults from a non-injurious stimulus to the skin, such as a lighttouch.

As used herein, the abbreviation “PWT” refers to paw withdrawalthreshold, a measurement of pain sensitivity in animals, in particular,rats and mice. Threshold is measured in terms of force. A reduction inthreshold suggests the development of allodynia.

As used herein, the term “nociceptor” refers to a specialized type ofnerve cell that senses and responds to pain.

As used herein, the term “hyperalgesia” refers to a condition of alteredperception such that stimuli which would normally induce a trivialdiscomfort cause significant pain. Hyperalgesia can be caused by damageto nociceptors present in tissue.

As used herein, the term “transgenic animal” refers to an animal whosegenome has been manipulated, such as an animal having DNA derived from adifferent organism, multiple copies of an endogeneous gene or having agene disruption.

As used herein, the term “wild-type animal” refers to an animal having anatural genome without any modifications. Wild-type animals are usuallyused as control animals in experiments using transgenic varieties of theanimal.

BRIEF DESCRIPTION OF THE FIGURES

The instant patent or application file contains at least one drawingexecuted in color. Copies of the patent or patent applicationpublication with color drawing(s) will be provided by the Office uponrequest and payment of the necessary fee.

FIGS. 1A-E show the results of experiments evidencing that ND2 is a Srcunique domain-interacting protein. FIG. 1A is a schematic diagramillustrating the domain structure of ND2, clones isolated from the yeasttwo-hybrid screen, and recombinant GST-tagged fusion proteins. FIG. 1Bshows a blot of ND2-GST fusion proteins probed with biotinylated Srcunique domain followed by a streptavidin-HRP conjugate. FIG. 1C shows ablot of ND2.1-GST probed with biotinylated domains of Src and Fynfollowed by streptavidin-HRP conjugate. FIG. 1D shows immunoblots ofco-immunoprecipitates from brain homogenate probed with anti-ND2,anti-Src or anti-Fyn. FIG. 1E shows an immunoblot ofco-immunoprecipitates from cultured Src^(+/+) and Src^(−/−) fibroblastsprobed with anti-ND2.

FIGS. 2A-E show the results of experiments evidencing that ND2 ispresent at the post-synaptic density. FIG. 2A shows immunoblots of PSDproteins probed with anti-ND2, anti-cytochrome c oxidase I (Cyto 1),anti-ND4, anti-PSD95, anti-NR1, anti-Src and anti-synaptophysin. FIG. 2Bshows immunoblots of mitochondrial proteins prepared by differentialcentrifugation probed with anti-ND2, anti-Cyto 1 and anti-ND4. FIG. 2Cshows immunoblots of PSD proteins showing the specificity of theN-terminal ND2 antibody by pre-adsorption with the antigenic peptideused to derive the antibody. FIG. 2D shows immunoblots of PSD andmitochondrial proteins probed with two independent rabbit polyclonalantibodies directed against two disparate regions of ND2. FIG. 2E showsthree representative post-embedding immunogold electron microscopyimages of rat hippocampus CA1 synapses, pre-synaptic. Scale bar is 200nm.

FIGS. 3A-B show the results of experiments evidencing that ND2 interactswith Src at the post-synaptic density. FIG. 3A shows immunoblots ofco-immunoprecipitates from PSD preparations probed with anti-ND2 oranti-Src. FIG. 3B shows recombinant ND2.1-GST fusion protein, but notND2.2-GST, ND2.3-GST, or GST alone, pulls Src from PSD preparations.

FIGS. 4A-G show the results of experiments evidencing that ND2 interactswith Src at the NMDAR complex. FIG. 4A shows immunoblots ofco-immunoprecipitates from PSD preparations probed with anti-ND2 or withanti-NMDA receptor subunit 1 (NR1). FIG. 4B shows an immunoblot ofco-immunoprecipitates from PSD preparations using anti-GluR2anti-GABA_(A)Rα, anti-GABA_(A)Rβ2/3 and anti-Kv3.1 antibodies toimmunoprecipitate. FIG. 4C shows a dot blot of ND2-GST fusion proteinsprobed with biotinylated Src40-58 or scrambled Src40-58 peptidesfollowed by streptavidin-HRP conjugate. FIG. 4D shows a blot ofND2.1-GST probed with boptinviated Src unique domain in the presence ofeither Src40-58 or scrambled Src40-58 peptides followed bystreptavidin-HRP conjugate. FIG. 4E shows immunoblots ofco-immunoprecipitates obtained from PSD proteins in the presence ofeither Src40-58 or scrambled Src40-58 probed with anti-ND2 or strippedand re-probed with anti-Src. FIG. 4F, left panel shows immunoblots ofco-immunoprecipitates obtained from PSD proteins in the presence ofeither Src40-58 or scrambled Src40-58. FIG. 4F, right panel showsimmunoblots of co-immunoprecipitates obtained from PSD proteins in thepresence of GST-ND2.1 fusion protein probed with anti-Src or anti-NR1.FIG. 4G shows immunoblots of co-immunoprecipitates obtained from PSDproteins in the presence of either Src40-58 or scrambled Src40-58peptides probed with anti-ND2 or stripped and re-probed with anti-NR1.

FIGS. 5A-F show the results of experiments evidencing that blockingexpression of ND2 prevents Src-dependent regulation of NMDA receptoractivity. FIG. 5A shows immunoblots of total soluble protein obtainedfrom cultured rat hippocampal neurons treated with 50 μg/mlchloramphenicol for 48 hours and probed with anti-ND2, anti-NR1 andanti-Src. FIG. 5B shows an immunoblot of co-immunoprecipitates obtainedfrom cultured hippocampal neurons, either treated or untreated with 50μg/ml chloramphenicol for 48 hours and probed with anti-NR1 or anti-Src.FIG. 5C shows summary histograms (left panel) of ATP level ormitochondrial membrane potential (ΔψM), as assessed by TMRM fluorescencedequenching (right panel), in cultured hippocampal neurons eitheruntreated or treated 50 μg/ml chloramphenicol for 48 hours. FIG. 5Dshows that the upregulation of NMDAR activity in the presence of the Srcactivator peptide EPO(pY)EEIPIA (SEQ ID NO:5), labeled as (pY)EEI (aminoacid residues 4-7 of SEQ ID NO:5), is prevented in neurons treated withchloramphenicol for 48 hours. FIG. 5E shows that the reduction of NMDAactivity in the presence of the Src40-58 peptide is also prevented inneurons treated with chloramphenicol for 48 hours. FIG. 5F shows asummary histogram of electrophysiology data. FIG. 5F shows amino acidresidues 4-7 of SEQ ID NO:5 (pY) EEI.

FIGS. 6A-C show the results of experiments evidencing that the Src40-49(SUDAPI-1) peptide specifically interacts with the ND2.1 peptide. FIG.6A is a schematic diagram depicting the Src40-58, Src40-49, Src49-58,and scrambled Src40-58 peptides. FIG. 6B shows the blot of the ND2.1-GSTfusion protein which was probed with biotinylated Src peptides followedby streptavidin-HRP conjugate. FIG. 6C shows the dot blots of ND2.1-GSTfusion proteins probed with biotinylated Src peptides followed bystreptavidin-HRP conjugate.

FIGS. 7A-D show results of experiments showing the effects of TSUDAPI-1on 2.5% formalin-induced flinching or biting/licking behaviors. FIG. 7Ashows the cumulative flinches in different phases observed within thehour. P1 represents a time period of 0-8 minutes; P2A represents a timeperiod of 12-28 minutes and P2B represents a time period of 32-60minutes. Values depict means (n=7, Src40-49Tat; n=20, saline). P<0.05,P<0.01 with student t test compared to saline control. FIG. 7B showsmeasurement of flinching behaviors observed within an hour. FIG. 7Cshows the cumulative biting/licking behaviors in different phasesobserved within the hour. FIG. 7D shows measurement of the time of eachbiting/licking behavior observed within an hour.

FIGS. 8A-D show results of experiments showing the effects of HIV-TAT on2.5% formalin-induced flinching or biting/licking behaviors. FIG. 8Ashows the cumulative flinches in different phases observed within thehour. P1 represents a time period of 0-8 minutes; P2A represents a timeperiod of 12-28 minutes and P2B represents a time period of 32-60minutes. Values depict means (n=7, HIV-Tat; n=20, saline). P<0.05,P<0.01 with student t test compared to saline control. FIG. 8B showsmeasurement of flinching behaviors observed within an hour. FIG. 8Cshows the cumulative biting/licking behaviors in different phasesobserved within the hour. FIG. 8D shows measurement of the time of eachbiting/licking behavior observed within an hour.

FIGS. 9A-B show SEQ ID NOS:6 and 7; FIG. 9A shows the nucleotidesequence encoding recombinant ND2.1 protein (SEQ ID NO:6); FIG. 9B showsthe amino acid sequence of recombinant ND2.1 protein (SEQ ID NO:7).

FIGS. 10A-B show immunoblots evidencing that ND2 and Src interact inmultiple, diverse tissues. FIG. 10A shows immunoblots ofco-immunoprecipitates from various tissues and FIG. 10B showsimmunoblots of co-immunoprecipitates from tissue homogenates probed withanti-ND2, anti-Src, or anti-Fyn. Tissues: B—brain; H—heart; I—intestine;K—kidney; Liv—liver; Lu—lung; P—pancreas; Sk—skeletal muscle; Sp—spleenand T—testis.

FIG. 11 shows graphs evidencing that intrathecal injection of 0.02 pmolof Src40-49TAT (TSUDAPI-1, SEQ ID NO:2) significantly reverses allodyniain rats.

FIG. 12 shows graphs evidencing that intravenous injection of 10 pmol/gof Src40-49TAT (TSUDAPI-1, SEQ ID NO:2) significantly reverses allodyniain rats.

FIG. 13 shows graphs evidencing that intravenous injection of 100 pmolSrc40-49TAT (TSUDAPI-1, SEQ ID NO:2) significantly reverses allodynia inwild type mice but does not further increase paw withdrawal threshold(PWT) in Src null mice.

FIGS. 14A-D show data illustrating the increase in tyrosinephosphorylation of the NR2B subunit after formalin injection(inflammatory pain model) and further show that this increase of NR2Btyrosine phosphorylation is significantly reduced by intrathecaladministration of Src40-49Tat (SEQ ID NO:2). FIG. 14A shows a westernblot of an immunoprecipitation using an anti-NR2B antibody and ananti-phosphorylated tyrosine antibody. FIG. 14B shows a graphquantifying the tyrosine phosphorylation calculated as a percent of thecontrol. This data was calculated prior to formalin injection and atthree times post-infection; at 5 minutes, 30 minutes and 60 minutes.FIG. 14C shows another western blot of an immunoprecipitation using ananti-NR2B antibody and an anti-phosphorylated tyrosine antibody. Thisblot evidences the reduction of formalin-induced tyrosinephosphorylation resulting from treatment with Src40-49Tat (SEQ ID NO:2)and the absence of reduction resulting from treatment with scrambledSrc40-49Tat (sSrc40-49Tat). FIG. 14D shows a graph quantifying thetyrosine phosphorylation (after treatment with Src40-49Tat or sSrc40-49)calculated as a percent of the control.

FIGS. 15A-B show data illustrating the increase in tyrosinephosphorylation of the NR2B subunit in animals having a cuff implant(neuropathic pain model) and further show that this increase of NR2Btyrosine phosphorylation is significantly reduced by intrathecaladministration of Src40-49Tat (SEQ ID NO:2). FIG. 15A shows a westernblot of an immunoprecipitation using an anti-NR2B antibody andanti-phosphorylated tyrosine antibody. FIG. 15B shows a graphquantifying the tyrosine phosphorylation calculated as a percent of thecontrol. This graph represents quantification of band (from westernblots) density in three experiments.

FIGS. 16A-E show the results of experiments evidencing that an antibodyagainst ND2 recognizes NADH-Ubiquinone Oxidoreductase 1 alpha subcomplex9 (NdufA9), a subunit of mitochondrial complex I (NCBI GeneID:4704).FIG. 16A shows immunoblots of PSD proteins probed with anti-ND2,anti-cytochrome c oxidase I (Cyto 1), anti-NdufA9, anti-PSD95, anti-NR1,anti-Src and anti-synaptophysin. FIG. 16B shows immunoblots ofmitochondrial proteins prepared by differential centrifugation probedwith anti-ND2, anti-Cyto 1 and anti-NdufA9. FIGS. 16C-E are identical toFIGS. 2C-E.

DETAILED DESCRIPTION OF THE INVENTION Example 1

NADH dehydrogenase subunit 2 (ND2) is a Src unique domain-bindingprotein.

A yeast two-hybrid screen of a fetal brain library using bait constructscontaining the murine Src unique domain was conducted in order to searchfor proteins that interact with the Src unique domain.

cDNAs encoding amino acids 4-82 (the Src unique domain) and amino acids4-150 (the Src unique and SH3 domains) of murine n-Src were ligated intopEG202 (Gyuris et al. Cell 75:791-803 1993) to create two expressionvectors encoding in frame LexA fusions containing the Src unique domain(the nucleotide sequence encoding Src is SEQ ID NO:3 and the amino acidsequence is SEQ ID NO:4). The bait constructs were then sequenced. Bothbaits were tested to ensure that the baits did not activatetranscription of the reporters in the absence of prey and that bothcould enter the nucleus and bind to LexA operators. To create theselection strains for screening, each bait plasmid was individuallytransformed into the yeast strain EGY48. EGY48 has an integrated Leu2selectable marker regulated by 6 LexA operator repeats, and carries areporter plasmid with the lacZ gene regulated by 8 LexA operatorrepeats. Bait-prey interactions that occur with low affinity result inactivation of the Leu2 reporter gene only, whereas high affinityinteractions result in activation of both the Leu2 and lacZ reportergenes, allowing for double selection of prey. The selection strain wastransformed with a representative activation-tagged cDNA prey fusionlibrary constructed using ˜1 kilobase EcoRI fragmented poly A(+) RNAfrom human fetal brain. Yeast transformed with the prey library(approximately 1.1×10⁶ clones) were screened by double selection onX-gal Leu⁻ medium. Prey cDNAs encoding proteins that interacted with thebait were isolated and sequenced.

Src, Fyn, and ND2 recombinant proteins were prepared. The cDNAs encodingthe SH3 and SH2 domains of mouse n-Src and Fyn were PCR subcloned,ligated in frame into pGEX4T-1 (Amersham Pharmacia Biotech, Baie d'Urfé,Québec), and sequenced. These plasmids, as well as plasmids encoding theunique domains of Src and Fyn in pGEX2T'6, were transformed into BL21bacteria, and GST fusion proteins were purified by glutathione affinitychromatography. To create the ND2.1, ND2.2, and ND2.3 GST fusionproteins, cDNAs encoding amino acids 239-321 (ND2.1-GST; SEQ ID NO:7),amino acids 189-238 (ND2.2-GST; SEQ ID NO:11), and amino acids 1-188(ND2.3-GST; SEQ ID NO:13) of human ND2 were PCR subcloned and ligatedinto pGEX4T-1 (the nucleotide sequence encoding ND2 is SEQ ID NO:8 andthe amino acid sequence is SEQ ID NO:9; the nucleotide sequencesencoding ND2.1; ND2.2 and ND2.3 are SEQ ID NOS:6, 10 and 12,respectively). Using PCR-based single nucleotide mutagenesis, all cDNAsencoding ND2 fusion proteins were corrected for differences betweenmitochondrial and nuclear codons to prevent premature translationtermination and protein truncation. All constructs were then confirmedby sequencing. The plasmids were transformed into bacteria, and GSTfusion proteins were purified by glutathione affinity chromatography.

Detailed protocols for in vitro binding assays, pull down assays,immunoblotting, and co-immunoprecipitation techniques can be found inPelkey et al. (Neuron 34:127-138 2002).

In two independent screens, cDNA fragments encoding overlapping regionswithin NADH dehydrogenase subunit 2 (ND2) were isolated (FIG. 1A). ND2is a 347 amino acid protein (SEQ ID NO:9) that is a subunit of the innermitochondrial membrane enzyme, NADH dehydrogenase (Complex I). ND2 isone of a group of seven oxidoreductase proteins that are encoded in themitochondrial genome and which co-assemble with 35 nuclear encodedsubunits to form Complex I. ND2 on its own lacks enzymatic activity (J.E. Walker Quarterly Reviews of Biophysics 25(3):253-324 1992; Sazanov etal. Journal of Molecular Biology 302:455-464 2000; Sazanov et al.Biochemistry 39:7229-7235 2000). FIG. 1A is a schematic diagramillustrating the domain structure of ND2, clones isolated from the yeasttwo hybrid screen, and recombinant GST-tagged fusion proteins. The linespoint out the beginning of the oxidoreductase domain at amino acidposition 23 and the end at amino acid position 197. Each clone andGST-fusion protein represent overlapping regions within ND2.

As yeast two-hybrid screening may reveal false positive protein-proteininteractions, the interaction between Src and ND2 was observed using anindependent methodological approach. Direct binding in vitro between ND2and Src was tested using recombinant proteins. A series of GST fusionproteins comprised of portions of ND2 that spanned the overlappingregion found with the yeast two-hybrid screen were made (FIG. 1A).Importantly, the cDNAs encoding each of the ND2 fusion proteins werecorrected for differences between mitochondrial and nuclear codons sothat the sequence of the ND2 portion of the fusion proteins was thatwhich would have been produced by translation in the mitochondria. Forexample, FIG. 9A shows the nucleotide sequence encoding recombinantND2.1 protein (SEQ ID NO:6). Codons that are highlighted with bold typewere altered by PCR-based single nucleotide mutagenesis. TGA was changedto TGG to prevent premature translation termination and proteintruncation. GAA was changed to GAG to remove a restriction enzyme site.Numbers in parenthesis correspond to equivalent positions in theendogenous human ND2 nucleotide sequence. FIG. 9B shows the amino acidsequence of recombinant ND2.1 protein (SEQ ID NO:7). Numbers inparenthesis correspond to equivalent positions in the endogenous humanND2 amino acid sequence. Each of the series of GST-fusion proteins wastested individually for interaction with the Src unique domain(“pull-down” assay). FIG. 1B shows a blot of ND2-GST fusion proteinsprobed with biotinylated Src unique domain followed by astreptavidin-HRP conjugate. A GST fusion protein containing amino acids239-321 of ND2 (ND2.1-GST; SEQ ID NO:7) was found that bound to theunique domain of Src (FIG. 1B). In contrast, GST fusion proteinscontaining amino acids 189-238 (ND2.2-GST) or 1-188 (ND2.3-GST) of ND2(ND2 protein sequence is SEQ ID NO:9) did not bind to the Src uniquedomain. These results, together with those from the yeast two-hybridscreen, indicate that ND2 is a Src unique domain-binding protein. Theresults indicate further that the Src-binding portion of ND2 iscontained within the region of amino acids 239-321 (SEQ ID NO:7). Thisregion of ND2 shows little conservation amongst the mitochondriallyencoded oxidoreductase proteins and is outside the so-called“oxidoreductase domain”, a signature region identified in allmitochondrially encoded subunits of NADH dehydrogenase (J. E. WalkerQuarterly Reviews of Biophysics 25(3):253-324 1992; Sazanov et al.Journal of Molecular Biology 302:455-464 2000; Sazanov et al.Biochemistry 39:7229-7235 2000) and some antiporters (Fearnley et al.Biochim. Biophys. Acta 1140:105-143 1992).

Another “pull-down” assay was conducted to determine whether the bindingof ND2 might generalize to other domains of Src or to other Src familytyrosine kinases.

However, it was found that ND2.1-GST did not bind to either of theprototypic protein-protein interaction domains of Src, the SH2 or SH3domains (FIG. 1C). FIG. 1C shows a blot of ND2.1-GST probed withbiotinylated domains of Src and Fyn followed by streptavidin-HRPconjugate.

To examine the potential interaction of ND2 with other kinases of theSrc family recombinant domains of Fyn were tested, the protein mostclosely related to Src but which has little primary sequenceconservation in the unique domain (Brown et al. Biochim. Biophys. Acta1287:121-149 1996; T. Pawson Nature 373:573-580 1995). It was found thatND2.1-GST did not interact in vitro with the Fyn unique domain; nor didND2.1 bind to the SH2 or SH3 domains of Fyn. Thus, the ND2.1 region doesnot interact with the SH2 or SH3 domains of Src or Fyn nor does itgenerally bind to the unique domain of Src family tyrosine kinases.

To investigate the possibility that Src and ND2 may interact in vivo,brain lysates were immunoprecipitated with antibodies directed againstND2 (anti-ND2) or against Src (anti-Src). It was found thatimmunoprecipitating Src led to co-immunoprecipitation of ND2 (FIG. 1D).FIG. 1D shows immunoblots of co-immunoprecipitates from brain homogenateprobed with anti-ND2, anti-Src or anti-Fyn as indicated. Non-specificIgG was used as a negative control for immunoprecipitation. Fyn wasreadily detected in the brain homogenate used as a starting material forthe co-immunoprecipitation (data not illustrated). Conversely,immunoprecipitating with anti-ND2 resulted in co-immunoprecipitation ofSrc. In contrast, anti-ND2 did not co-immunoprecipitate Fyn and neitherND2 nor Src was immunoprecipitated with a non-specific IgG (FIG. 1D). Asan independent immunoprecipitation control it was found that ND2 wasco-immunoprecipitated by anti-Src from Src^(+/+) fibroblasts but notfrom Src^(−/−) fibroblasts (FIG. 1E). FIG. 1E shows an immunoblot ofco-immunoprecipitates from cultured Src^(+/+) and Src^(−/−) fibroblastsprobed with anti-ND2. Non-specific IgG was used as a negative controlfor immunprecipitation, and immunoblotting of ND2 protein from both celllines was used as a positive control. Thus, in addition to finding theND2-Src unique domain interaction in two yeast two-hybrid screens and invitro binding assays with recombinant proteins, it was found that ND2and Src co-immunoprecipitated with each other, which together led to theconclusion that the ND2 is a Src unique-domain binding protein that mayinteract with Src in vivo.

Example 2

ND2 is present in post-synaptic densities in brain.

Post-synaptic density proteins (Kennedy et al. Proceedings of theNational Academy of Science USA 80:7357-7361 1983) were prepared fromrat brain as described in detail (Pelkey et al. Neuron 34:127-138 2002).Cellular fractionation of rat brain tissue into nuclear, heavymitochondrial, light mitochondrial, microsomal, and cytosolic fractionswas performed by differential centrifugation of tissue homogenate in0.25 M sucrose/10 mM HEPES-NaOH, 1 mM EDTA, pH 7.4 with 2 μg each ofaprotinin, pepstatin A, and leupeptin (Sigma, St. Louis, Mo.) at 4° C.Nuclei were pelleted by centrifugation at 1 000 g for 10 minutes, thesupernatant was removed and spun at 3 000 g for 10 minutes to obtain aheavy mitochondrial pellet. The supernatant was removed and spun at 16000 g for 15 minutes to obtain a light mitochondrial pellet. Thesupernatant was removed and spun at 100 000 g for 1 hour to obtain amicrosomal pellet and the cytosolic fraction. All pellets were thenresuspended in RIPA buffer (50 mM Tris pH 7.6, 150 mM NaCl, 1 mM EDTA,1% NP-40, 2.5 mg/ml NaDOC, 1 mM Na₃VO₄ 1 mM PMSF, and 2 μg/ml each ofprotease inhibitors). The light mitochondrial fraction was used insubsequent experiments. For Western blots, 50 μg of total protein wasloaded per lane, resolved by SDS-PAGE, transferred to nitrocellulosemembranes, and probed with anti-ND2, anti-Cytol and anti-ND4 (mousemonoclonals, Molecular Probes Inc., Eugene, Oreg.), anti-PSD95 (mousemonoclonal clone 7E3-1B8, Oncogene Research Products, Cambridge, Mass.),anti-NR1 (mouse monoclonal clone 54.1, Pharmingen), anti-Src, oranti-synaptophysin (mouse monoclonal, Sigma).

Post-embedding immunogold electron microscopy was carried out. SpragueDawley rats were anesthetized and perfused with 4% paraformaldehyde plus0.5% glutaraldehyde in 0.1 M phosphate buffer. Parasagittal sections ofthe hippocampus were cryoprotected in 30% glycerol and frozen in liquidpropane. Frozen sections were immersed in 1.5% uranyl acetate inmethanol at −90° C., infiltrated with Lowicryl HM-20 resin at −45° C.,and polymerized with ultraviolet light. Sections were incubated in 0.1%sodium borohydride plus 50 mM glycine in TBS and 0.1% Triton X-100(TBST), followed by 10% normal goat serum (NGS) in TBST, primaryantibody in 1% NGS in TBST, and immunogold (10 nm; Amersham PharmaciaBiotech) in 1% NGS in TBST plus 0.5% polyethylene glycol. Finally, thesections were stained in uranyl acetate and lead citrate prior toanalysis.

In the CNS a prominent subcellular location for Src is in thepost-synaptic density (PSD) (Yu et al. Science 275:674-678 1997), asubsynaptic specialization at glutamatergic synapses comprised ofα-amino-3-hydroxy-5-methylisoxazolepropionic acid (AMPA-) and NMDA-typeglutamate receptors together with scaffolding, signaling and regulatoryproteins (Walikonis et al. Journal of Neuroscience 20:4069-4080 2000).Because Src is known to regulate subsynaptic NMDARs (Yu et al. Science275:674-678 1997), if ND2 is the protein mediating the interactionbetween NMDARs and the unique domain of Src then ND2 is predicted to bepresent in the PSD. This was tested by preparing PSD proteins from ratbrain homogenates by sequential fractionation and determining whetherND2 was present in this fraction. Characteristic of a bona fide PSDfraction, the fraction which was prepared contained post-synapticproteins including PSD-95 and NMDA receptor subunit proteins but lackedthe pre-synaptic protein synaptophysin (FIG. 2A). FIG. 2A showsimmunoblots of PSD proteins probed with anti-ND2, anti-cytochrome coxidase I (Cyto 1), anti-ND4, anti-PSD95, anti-NR1, anti-Src andanti-synaptophysin as indicated. It was found that ND2 was present inthe PSD fraction and the amount of ND2 estimated in this fraction wasapproximately 15% of that in the total brain homogenate. In contrast toND2, neither the oxidoreductase protein ND4, anothermitochondrially-encoded component of Complex I (J. E. Walker QuarterlyReviews of Biophysics 25(3):253-324 1992; Sazanov et al. Journal ofMolecular Biology 302:455-464 2000; Sazanov et al. Biochemistry39:7229-7235 2000) nor cytochrome c oxidase subunit 1 (Cyto 1), an innermitochondrial membrane protein that is part of Complex IV (Marusich etal. Biochim. Biophys. Acta 1362:145-159 1997), was detectable in the PSDfraction. On the other hand, Cyto 1 and ND4, as well as ND2, werereadily detected in proteins from brain mitochondria (FIG. 2B).Subsequent investigation indicated that the NdufA9 (NADH-ubiquinoneoxidoreductase 1 alpha subcomplex 9) subunit of mitochondrial complex Iwas detected and not ND4 (FIG. 16B). FIG. 2B shows immunoblots ofmitochondrial proteins prepared by differential centrifugation probedwith anti-ND2, anti-Cyto 1 and anti-ND4. Neither NR1 nor NR2A/B wasdetected in the mitochondrial fraction (data not shown).

As noted above, subsequent investigations indicated that the antibodyinitially thought to recognize the mitochondrial protein ND4, a controlin the study, actually recognizes NADH-Ubiquinone Oxidoreductase 1 alphasubcomplex 9 (NdufA9). Like ND4, NdufA9 protein has a molecular weightof 39 kDa and is a subunit of NADH dehydrogenase (mitochondrial complexI). However, unlike ND4, NdufA9 is encoded in the nucleus. BecauseNdufA9 is a subunit of mitochondrial complex I, as is ND4, NdufA9 isalso an appropriate control for the instantly described experiments(Gingrich et al. PNAS 103(25):9744 2006; published online on Jun. 8,2006). Referring now to FIGS. 16A-B, characteristic of a bona fide PSDfraction, the fraction which was prepared contained post-synapticproteins including PSD-95 and NMDA receptor subunit proteins but lackedthe pre-synaptic protein synaptophysin (FIG. 16A) FIG. 16A showsimmunoblots of PSD proteins probed with anti-ND2, anti-cytochrome coxidase I (Cyto 1), anti-NdufA9, anti-PSD95, anti-NR1, anti-Src andanti-synaptophysin as indicated. It was found that ND2 was present inthe PSD fraction. In contrast to ND2, neither the NADH-UbiquinoneOxidoreductase 1 alpha subcomplex 9 (NdufA9), a subunit of mitochondrialcomplex I, nor the cytochrome c oxidase subunit 1 (Cyto 1), an innermitochondrial membrane protein that is part of Complex IV (Marusich etal. Biochim. Biophys. Acta 1362:145-159 1997), were detectable in thePSD fraction. On the other hand, Cyto 1 and NdufA9, as well as ND2, werereadily detected in proteins from brain mitochondria (FIG. 16B). FIG.16B shows immunoblots of mitochondrial proteins prepared by differentialcentrifugation probed with anti-ND2, anti-Cyto 1 and anti-NdufA9. FIGS.16C-E are identical to FIGS. 2C-E.

Although the molecular size of the protein detected by anti-ND2 in thePSD preparation matched that of ND2 in mitochondria, it is conceivablethat the protein detected in the PSD preparation was not ND2 but aprotein of the same molecular size that was recognized by anti-ND2.However, it was found that incubating anti-ND2 with the antigen to whichthe antibody was raised prevented the immunoblotting signal (FIG. 2C).FIG. 2C shows immunoblots of PSD proteins showing the specificity of theN-terminal ND2 antibody by pre-adsorption with the antigenic peptideused to derive the antibody. Morever, it was found that a separateantibody directed towards a distinct epitope in a region of ND2 remotefrom that of the anti-ND2 epitope also detected ND2, at the correctmolecular size, in the PSD preparation, as well as in the mitochondrialpreparation (FIG. 2D). FIG. 2D shows immunoblots of PSD andmitochondrial proteins probed with two independent rabbit polyclonalantibodies directed against two disparate regions of ND2. The N-terminalND2 antibody was used for all subsequent experiments illustrated. Thus,ND2 was found in the PSD preparation by two separate antibodies, andthis could not be accounted for by a general contamination withmitochondrial proteins because neither Cyto 1 nor ND4 were detected inthe PSD.

In addition to examining PSD protein preparations, the presence of ND2in PSDs was tested for by means of post-embedding immunogold electronmicroscopy in the CA1 stratum radiatum of rat hippocampus (Petralia etal. Nature Neuroscience 2:31-36 1999; Sans et al. Journal ofNeuroscience 20:1260-1271 2000). With this experimental approach thetissue is fixed immediately after the animal is sacrificed and prior tosectioning so that protein localization is preserved. ND2 labeling wasfound, as visualized by secondary antibody conjugated to 10 nm goldparticles, in the PSD and the postsynaptic membrane in dendritic spinesof CA1 neurons (FIG. 2E), as well as over mitochondria (notillustrated). FIG. 2E shows three representative post-embeddingimmunogold electron microscopy images of rat hippocampus CA1 synapses,pre-synaptic. Scale bar is 200 nm. ND2 labeling was enriched in thepost-synaptic membrane approximately 30-fold as compared with the plasmamembrane in the remainder of the dendritic spine (0.37 particles perPSD/section versus 0.012, p<0.05) and there was no obvious accumulationof ND2 labeling along the plasma membrane of the dendritic shaft. TheND2 labeling observed in the PSD and post-synaptic membrane could nothave been due to labeling in mitochondria because it is known thatmitochondria are excluded from dendritic spines (Shepherd et al. Journalof Neuroscience 18(20):8300-8310 1998). Thus, these results indicatethat ND2 is present in the biochemically defined PSD protein fractionand is localized at PSDs in CA1 neurons.

Example 3

ND2 interacts with Src at the NMDA receptor complex in post-synapticdensities.

Since previous results indicate that ND2 is present in PSDs from brain,it was examined whether ND2 interacts with Src in PSDs. It was foundthat immunoprecipitating ND2 from the PSD fraction led toco-immunoprecipitation of Src and vice versa (FIG. 3A), indicating thatND2 and Src interact post-synaptically at glutamatergic synapses. FIG.3A shows immunoblots of co-immunoprecipitates from PSD preparationsprobed with anti-ND2 or anti-Src as indicated. Non-specific IgG (eitherrabbit or mouse) was used as a negative control for both antibodies.Moreover, Src was pulled from the PSD fraction by the fusion proteinND2.1-GST, but not by either ND2.2- or ND2.3-GST (FIG. 3B). FIG. 3Bshows recombinant ND2.1-GST fusion protein, but not ND2.2-GST,ND2.3-GST, or GST alone, pulls Src from PSD preparations. Thus, as itwas found with the Src-ND2 binding in vitro, these results indicate thatamino acids 239-321 of ND2 (SEQ ID NO:7) are both necessary andsufficient for ND2 to interact with Src in the PSD.

The hypothesis that ND2 is the protein mediating the interaction betweenSrc and NMDARs requires that, in addition to being present in the PSDand interacting there with Src, ND2 is part of NMDAR complex ofproteins. To determine whether ND2 is a component of the NMDAR proteincomplex, NMDAR complexes were immunoprecipitated from the PSD fraction,using an antibody directed against the core NMDAR subunit NR1(Dingledine et al. Pharmacology Reviews 51:7-61 1999), and theco-immunoprecipitating proteins were probed with anti-ND2. It was foundthat ND2 co-immunoprecipitated (FIG. 4A), and conversely,immunoprecipitating with anti-ND2 led to co-immunoprecipitation of NR1(FIG. 4A). FIG. 4A shows immunoblots of co-immunoprecipitates from PSDpreparations probed with anti-ND2 or with anti-NMDA receptor subunit 1(NR1) as indicated. Non-specific IgG (either rabbit or mouse) was usedas a negative control for both antibodies. Neither ND2 nor NR1 wasimmunoprecipitated by non-specific IgG, and ND2 did notco-immunoprecipitate with the potassium channel Kv3.1 (FIG. 4B), anegative control for non-specific immunoprecipitation of post-synapticproteins, therefore it was concluded that ND2 is an NMDAR complexprotein. FIG. 4B shows an immunoblot of co-immunoprecipitates from PSDpreparations using anti-GluR2, anti-GABA_(A)Rα, anti-GABA_(A)Rβ2/3 andanti-Kv3.1 antibodies to immunoprecipitate. Probe was anti-ND2.Importantly, neither ND4 nor Cyto 1 was detected inco-immunoprecipitates of NR1 (not illustrated) indicating thatmitochondrial proteins in general are not components of the NMDARcomplex. Moreover, ND2 did not co-immunoprecipitate with GluR2,GABA_(A)Rα or GABA_(A) Rβ2/3 (FIG. 4B) indicating that ND2 is not adetectable component of AMPA receptor or γ-aminobutyric acid (GABA)receptor complexes.

Thus, while ND2 is a component of NMDAR complexes it is not generally acomponent of neurotransmitter receptor complexes in the brain.

Example 4

ND2 acts as an adapter protein for Src.

Src40-58 and scrambled Src peptides were biotinylated by incubating withSulfo-NHS-Biotin (Pierce Chemical Co., Rockford, Ill.) for 30 minutes atroom temperature (SEQ ID NO:4, Src protein). The biotinylation reactionwas then quenched by the addition of Tris-HCl (pH 8.0) to a finalconcentration of 20 mM. Purified recombinant fusion proteins (˜20 μgeach) were dotted onto nitrocellulose and dried overnight. Membraneswere blocked with 5% BSA in PBS for 1 hour, after which biotinylatedpeptides (30 μg/ml) diluted 1:1000 in fresh 5% BSA in PBS were added.The membranes were incubated with the peptides for 1 hour, washed, andprobed using a streptavidin-HRP conjugate. Bound probe was then detectedon film using an ECL kit.

ND2 acts as an adapter protein for Src. Amino acids 40-58 (SEQ ID NO:4)within the Src unique domain have been implicated in the binding of Srcto the interacting protein in the NMDAR complex (Yu et al. Science275:674-678 1997; Lu et al. Science 279:1363-1368 1998; Yu et al. Nature396:469-474 1998) and thus, ND2 was predicted to bind to this region ofSrc. This prediction was examined in vitro using a peptide with thesequence of amino acids 40-58 (Src40-58; SEQ ID NO:4) which was found tobind directly to ND2.1-GST (FIG. 4C) in vitro. In contrast, a peptidewith identical amino acid composition, but a scrambled sequence(scrambled Src40-58), did not bind to ND2.1-GST. Neither Src40-58 norscrambled Src40-58 bound to ND2.2-GST, ND2.3-GST or to GST alone (FIG.4C). FIG. 4C shows a dot blot of ND2-GST fusion proteins probed withbiotinylated Src40-58 or scrambled Src40-58 peptides followed bystreptavidin-HRP conjugate. Furthermore, the effect of Src40-58 on theinteraction between Src and ND2 was examined (FIGS. 4D and 4E). It wasfound that incubating ND2.1-GST with Src40-58 prevented this fusionprotein from pulling down the Src unique domain protein in vitro (FIG.4D). FIG. 4D shows a blot of ND2.1-GST probed with boptinylated Srcunique domain in the presence of either Src40-58 or scrambled Src40-58peptides followed by streptavidin-HRP conjugate. On the other hand,scrambled Src40-58 did not affect the interaction between the ND2.1-GSTand Src unique domain proteins. Incubating PSD proteins with Src40-58prevented the co-immunoprecipitation of ND2 by anti-Src but this was notaffected by scrambled Src40-58 (FIG. 4E). FIG. 4E shows immunoblots ofco-immunoprecipitates obtained from PSD proteins in the presence ofeither Src40-58 or scrambled Src40-58 probed with anti-ND2 or strippedand re-probed with anti-Src. Importantly, Src40-58 did not affect theimmunoprecipitation of Src from PSDs. Thus, it was concluded that aminoacids 40-58 of Src interact with the region spanned by ND2.1, therebymediating the binding between the Src unique domain and ND2.

As ND2 alone is not catalytically active (J. E. Walker Quarterly Reviewsof Biophysics 25(3):253-324 1992; Sazanov et al. Journal of MolecularBiology 302:455-464 2000; Sazanove et al. Biochemistry 39:7229-72352000), its functional role in the NMDAR complex was investigated. ND2might be a phosphorylation target for Src, but it was found that ND2immunoprecipitated from PSD protein fractions was not detectablyphosphorylated on tyrosine. Moreover, inclusion of ND2.1-GST did notalter the catalytic activity of Src in vitro (not illustrated)consistent with the binding of ND2 to the unique domain rather than tothe regulatory or catalytic domains. Thus, it is unlikely that ND2 is atarget of Src or a regulator of Src kinase activity.

However, it was found that the co-immunoprecipitation of Src with NMDARs(FIG. 4F, left panel) was suppressed by Src40-58, but not scrambledSrc40-58, and by ND2.1 (FIG. 4F, right panel) indicating that theassociation of Src with the NMDAR complexes depends on the interactionwith ND2. FIG. 4F, left panel shows immunoblots of co-immunoprecipitatesobtained from PSD proteins in the presence of either Src40-58 orscrambled Src40-58. FIG. 4F, right panel shows immunoblots ofco-immunoprecipitates obtained from PSD proteins in the presence ofGST-ND2.1 fusion protein probed with anti-Src or anti-NR1 as indicated.In contrast, the co-immunoprecipitation of ND2 with NMDARs was notaffected by Src40-58 (FIG. 4G), implying that binding ND2 to Src is notnecessary for ND2 to associate with NMDAR complexes. FIG. 4G showsimmunoblots of co-immunoprecipitates obtained from PSD proteins in thepresence of either Src40-58 or scrambled Src40-58 peptides probed withanti-ND2 or stripped and re-probed with anti-NR1. Taking these resultstogether, it was concluded that ND2 may function as an adapter proteinthat anchors Src in the NMDAR complex.

Example 5

Loss of ND2 in neurons prevents the regulation of NMDA receptor activityby Src.

Fetal rat hippocampal neurons were prepared, cultured, and used forelectrophysiological recordings 12-17 days after plating. Methods forwhole cell recordings are described in Pelkey et al. (Neuron 34:127-1382002).

It was hypothesized that if ND2 is a Src adapter protein then loss ofND2 should prevent the upregulation of NMDAR activity by endogenous Src(Yu et al. Science 275:674-678 1997). This was tested by investigatingminiature excitatory post-synaptic currents (mEPSCs) recorded fromcultured hippocampal neurons (MacDonald et al. Journal of Physiology(London) 414:17-34 1989). In these neurons the NMDAR-mediated componentof mEPSCs is increased by activating endogenous Src with a high-affinityactivating phosphopeptide EPQ(pY)EEIPIA (Liu et al. Oncogene 8:1119-11261993) and is reduced by applying Src40-58 (Yu et al. Science 275:674-6781997). It is predicted that each of these effects will be lost byblocking the expression of ND2, if it acts as an adapter protein for Srcin the NMDAR complex. In order to suppress ND2 expression, thehippocampal cultures were treated with chloramphenicol to selectivelyinhibit translation of mitochondrially encoded proteins but nottranslation of proteins encoded in the nucleus (Ibrahim et al. Journalof Biological Chemistry 251:108-115 1976). After 48 hours treatment withchloramphenicol it was found that the level of ND2 in the cultures wasreduced by more than 95% whereas there was no significant change in thelevels of the nuclear encoded proteins examined (FIG. 5A). FIG. 5A showsimmunoblots of total soluble protein obtained from cultured rathippocampal neurons treated with 50 μg/ml chloramphenicol for 48 hoursand probed with anti-ND2, anti-NR1 and anti-Src as indicated.Importantly, chloramphenicol did not affect the level of Src or of theNMDAR subunit NR1 but did suppress the co-immunoprecipitation of Srcwith the NMDAR complex (FIG. 5B), as predicted if ND2 is an adapterprotein linking Src to the complex. FIG. 5B shows an immunoblot ofco-immunoprecipitates obtained from cultured hippocampal neurons, eithertreated or untreated with 50 μg/ml chloramphenicol for 48 hours andprobed with anti-NR1 or anti-Src.

The effect of the 48 hours treatment with chloramphenicol on the ATPlevels, mitochondrial membrane potential, viability and generalfunctioning of the hippocampal neurons in culture was examined. It wasfound that chloramphenicol did not significantly affect the level of ATPlevels in the cultures (FIG. 5C), consistent with the lack of effect ofchloramphenicol treatment for up to 55 hours on ATP levels in other celltypes in culture (Ramachandran et al. Proceedings of the NationalAcademy of Science USA 99:6643-6648 2002). FIG. 5C shows summaryhistograms (left panel) of ATP level or mitochondrial membrane potential(ΔψM), as assessed by TMRM fluorescence dequenching (right panel), incultured hippocampal neurons either untreated or treated 50 μg/mlchloramphenicol for 48 hours. To examine the effect of chloramphenicolon mitochondrial membrane potential (ΔψM) in individual neurons, thedequenching of the potentiometric fluorescent cationic dyetetramethylrhodamine methyl ester (TMRM) by the mitochondrial uncouplercarbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) was monitored(Reers et al. Biochemistry 30:4480-4486 1991). The dequenching responseevoked by bath-applied FCCP (2 μM) in neurons fromchloramphenicol-treated or control cultures was assessed. It was foundthat the dequenching response of chloramphenicol-treated neurons was notdifferent from that of untreated neurons (FIG. 5C), indicating that ΔψMwas not affected by chloramphenicol. Moreover, it was found that neuronstreated with chloramphenicol were not distinguishable from untreatedneurons in terms of cell number, gross morphology, resting membranepotential, resting intracellular calcium concentration, action potentialamplitude, or mEPSC frequency (data not illustrated). Thus, from thesedata together it was concluded that treatment with chloramphenicol for48 hours did not detectably compromise the functioning of the neurons.Nevertheless, it was noted that the intracellular solution used for allwhole-cell recordings contained 2 mM Mg-ATP, so that the level ofintracellular ATP was equal in all cells throughout the experiments.

In neurons treated with chloramphenicol for 48 hours it was found thatthe NMDAR component of the mEPSCs was not affected by administeringeither the EPQ(pY)EEIPIA (SEQ ID NO:5) peptide or the Src40-58 peptide(FIGS. 5D-F). In contrast, in control experiments administeringEPQ(pY)EEIPIA (SEQ ID NO:5) increased the NMDAR component of mEPSCs by172±28% and application of Src40-58 decreased the NMDAR component to56±4% (FIGS. 5D-F). Chloramphenicol was present during the recordingperiods of the control experiments and therefore the loss of effect ofthe EPQ(pY)EEIPIA (SEQ ID NO:5) and Src40-58 peptides cannot beattributed to an acute effect of chloramphenicol. FIG. 5D shows that theupregulation of NMDAR activity in the presence of the Src activatorpeptide EPQ(pY)EEIPIA (SEQ ID NO:5), labeled as (pY)EEI (amino acidresidues 4-7 of SEQ ID NO:5), is prevented in neurons treated withchloramphenicol for 48 hours. FIG. 5E shows that the reduction of NMDAactivity in the presence of the Src40-58 peptide is also prevented inneurons treated with chloramphenicol for 48 hours. Composite traces areshown in black, the NMDAR component in dark grey, and the AMPARcomponent in light grey. Scale bars are 50 ms/10 pA. FIG. 5F shows asummary histogram of electrophysiology data. NMDA component data werecalculated as Q_(20′)/Q_(2′), and AMPA component data were calculated asA_(20′)/A_(2′). A 48 hour chloramphenicol treatment prevents themodulation of NMDAR function by the Src activator peptide (SEQ ID NO:5)and Src40-58 peptides, while neither of these reagents affected the AMPAreceptor component of the MEPSCs under the recording conditions used.An * indicates a significant difference, Student's t-test, p<0.05.Taking our results together, it is concluded that Src-dependentregulation of the activity of NMDARs depends on expression of ND2through its anchoring of Src to the NMDAR complex.

Example 6

Src40-49 interacts directly with ND2

To detect the binding of ND2.1-GST with Src peptides, the ND2.1-GSTfusion protein was purified on glutathione SEPHAROSE. Src40-58,Src40-49, Src49-58, and scrambled Src40-58 peptides (30 mg/ml;synthesized by HSC Peptide Synthesis Facility; all four peptides areschematically depicted in FIG. 6A) were biotinylated by incubating withSulfo-NHS-Biotin (Pierce Chemical Co., Rockford, Ill.) for 30 minutes atroom temperature. The biotinylation reaction was then quenched by theaddition of Tris-HCl (pH 8.0) to a final concentration of 20 mM.Biotinylated peptides were incubated with ND2.1-GST on beads for 1 hourat 4° C. The beads were washed three times with PBS/0.1% Triton X-100,then resuspended in PBS+SDS-PAGE sample buffer. After briefcentrifugation, samples were resolved by SDS-PAGE, transferred tonitrocellulose membranes, and probed using a streptavidin-HRP conjugate(Sigma, St. Louis, Mo.). Bound probe was then detected on film using anECL kit (Amersham Pharmacia Biotech, Baie d'Urfé, Québec). FIG. 6B showsthe blot of the ND2.1-GST fusion protein which was probed withbiotinylated Src peptides followed by streptavidin-HRP conjugate.

Src40-58, Src40-49, Src49-58, scrambled Src40-58, TAT-Src40-49, andscrambled TAT-Src40-49 peptides were biotinylated by incubating withSulfo-NHS-Biotin (Pierce Chemical Co., Rockford, Ill.) for 30 minutes atroom temperature. The biotinylation reaction was then quenched by theaddition of Tris-HCl (pH 8.0) to a final concentration of 20 mM.Purified recombinant fusion proteins (˜20 μg each) were dotted ontonitrocellulose and dried overnight. Membranes were blocked with 5% BSAin PBS (pH 7.5) for 1 hour, after which biotinylated peptides (30 μg/ml)diluted 1:1000 in fresh 5% BSA in PBS were added. The membranes wereincubated with the peptides for 1 hour, washed, and probed withstreptavidin-HRP conjugate. Bound probe was then detected on film usingan ECL kit. FIG. 6C shows the dot blots of ND2.1-GST fusion proteinsprobed with biotinylated Src peptides followed by streptavidin-HRPconjugate.

Example 7

TAT-Src40-49 (TSUDAPI-1) reduces pain behavior

Male Sprague-Dawley rats 150-200 g were used for all experiments. Ratswere housed in pairs, maintained on a 12/12 hour light/dark cycle, andallowed free access to food and water. All experiments were conductedduring 10 am and 5 μm.

Peptide Src40-49Tat (TSUDAPI-1; SEQ ID NO:2) or Tat alone (amino acidresidues 1-11 of SEQ ID NO:2) was dissolved in sterilized saline.Peptide or saline was injected intravenously at a volume 1 ml/Kg intorat's tail 45 minutes before behavioral testing. Injections were doneunder brief halothane anesthesia and rats were returned to the cagesafter injections.

The formalin test was performed as previously described (Liu et al.European Journal of Pharmacology 408(2):143-152 2000). Rats were placedin a plexiglass observation chamber for an initial 20 minutes to allowacclimatization to the testing environment. Formalin 2.5% was injectedsubcutaneously in a volume of 50 ml into the plantar aspect of the hindpaw. Following injections, rats were returned to the observation chamberand monitored for flinching behaviors (lifting, shaking and overtflinching with a ripple over the haunch) and biting/licking time. Tworats in adjacent chambers were observed at one time, with observationsoccurring in alternate 2 minute bins. Recorded episodes were notcorrected, thus values represent about half of the total behaviorsexpressed.

FIGS. 7A-D show the effect of Src40-49Tat (0.1 pmol) on 2.5% formalininduced flinching or biting/licking behaviors. Peptides or salinecontrols were injected 45 minutes before behavioral testing. FIG. 7Bshows measurement of flinching behaviors observed within an hour. FIG.7A shows the cumulative flinches in different phases observed within thehour. P1 represents a time period of 0-8 minutes; P2A represents a timeperiod of 12-28 minutes and P2B represents a time period of 32-60minutes. Values depict means (n=7, Src40-49Tat; n=20, saline). P<0.05,P<0.01 with student t test compared to saline control. FIG. 7D showsmeasurement of the time of each biting/licking behavior observed withinan hour. FIG. 7C shows the cumulative biting/licking behaviors indifferent phases observed within the hour. P1 represents a time periodof 0-8 minutes; P2A represents a time period of 12-28 minutes and P2Brepresents a time period of 32-60 minutes. Values depict means (n=7,Src40-49Tat; n=20, saline). P<0.05, P<0.01 with student t test comparedto saline control.

FIGS. 8A-D show the effect of HIV-Tat (1 pmol/g) on 2.5% formalininduced flinching or biting/licking behaviors. Peptides or salinecontrols were injected 45 minutes before behavioral testing. FIG. 8Bshows measurement of flinching behaviors observed within an hour. FIG.8A shows the cumulative flinches in different phases observed within thehour. P1 represents a time period of 0-8 minutes; P2A represents a timeperiod of 12-28 minutes and P2B represents a time period of 32-60minutes. Values depict means (n=7, HIV-Tat; n=20, saline). P<0.05,P<0.01 with student t test compared to saline control. FIG. 8D showsmeasurement of the time of each biting/licking behavior observed withinan hour. FIG. 8C shows the cumulative biting/licking behaviors indifferent phases observed within the hour. P1 represents a time periodof 0-8 minutes; P2A represents a time period of 12-28 minutes and P2Brepresents a time period of 32-60 minutes. Values depict means (n=7,HIV-Tat; n=20, saline). P<0.05, P<0.01 with student t test compared tosaline control. As compared to HIV-Tat alone and the saline control, theSrc40-49Tat peptide is shown to reduce pain behaviors over a time periodof an hour.

It is known that tyrosine phosphorylation of the NR2 subunits plays akey role in NMDA receptor activation (Moon et al. PNAS USA 91:3954-39581994; Lau et al. Journal of Biological Chemistry 270:20036-20041 1995;Xiong et al. Journal of Neuroscience 19:RC37(1-6) 1999). It is alsoknown that inflammatory hyperalgesia is associated with rapid andprolonged enhancement of tyrosine phosphorylation of the NR2B subunitsof NMDA receptors (Guo et al. The Journal of Neuroscience22(14):6208-6217 2002). Thus, considering that protein phosphorylationis a major mechanism for both normal and pathological receptor function,the effect of the formalin test on receptor phosphorylation wasexamined.

FIGS. 14A-D illustrate the increase in tyrosine phosphorylation of theNR2B subunit after formalin injection (FIGS. 14A-B) and furtherillustrate that this increase of NR2B tyrosine phosphorylation issignificantly reduced by intrathecal (i.t.) administration ofSrc40-49Tat (SEQ ID NO:2) (FIGS. 14C-D). FIG. 14A shows a western blotof an immunoprecipitation using an anti-NR2B antibody and ananti-phosphorylated tyrosine antibody. It can be seen that by 60 minutespost-injection of formalin, tyrosine phosphorylation of the NR2B subunitincreases. FIG. 14B shows a graph quantifying the tyrosinephosphorylation calculated as a percent of the control. This data wascalculated prior to formalin injection and at three timespost-injection; at 5 minutes, 30 minutes and 60 minutes. As can be seen,the amount of NR2B subunit that is phosphorylated peaks at around 30minutes post-injection. FIG. 14C shows another western blot of animmunoprecipitation using an anti-NR2B antibody and ananti-phosphorylated tyrosine antibody. This blot evidences the reductionof formalin-induced tyrosine phosphorylation resulting from treatmentwith Src40-49Tat (SEQ ID NO:2) and the absence of reduction resultingfrom treatment with scrambled Src40-49Tat (sSrc40-49Tat). FIG. 14D showsa graph quantifying the tyrosine phosphorylation (after treatment withSrc40-49Tat or sSrc40-49) calculated as a percent of the control. Thisdata was calculated at 60 minutes post-injection of formalin to treatedanimals.

Example 8

ND2-Src interaction in multiple tissues

Total soluble protein was prepared from pre-weighed rat tissues byhomogenization at 4° C. in 0.25 M sucrose/10 mM HEPES-NaOH, 1 mM EDTA,pH 7.4 with 2 μg/ml each of aprotinin, pepstatin A, and leupeptin.Following brief configuration of the samples at 4 000 g, NP-40 was addedto 1% (vol/vol) to the cleared supernatants. After incubation for 10minutes, the protein concentration of the samples was determined bydetergent compatible protein assay (BioRad Laboratories, Mississauga,Ontario) and equilibrated. The solubilized proteins were centrifugedbriefly at 14 000 g to remove insoluble material and then incubated with5 μg of either anti-ND2 (rabbit polyclonal from Dr. R. F. Doolittle,UCSD, CA; described in Mariottini et al. PNAS USA 83:1563-1567 1986),anti-Src (mouse monoclonal clone 327 from J. Bolen, DNAX, Palo Alto,Calif.) or control, non-specific rabbit or mouse IgG (Sigma) overnightat 4° C. Immune complexes were isolated by the addition of 10 μl ofprotein G-SEPHAROSE beads followed by incubation for 2 hours at 4° C.Immunoprecipitates were then washed three times with RIPA buffer,re-suspended in RIPA buffer+SDS-PAGE sample buffer and boiled for 5minutes. The samples were resolved by SDS-PAGE, transferred tonitrocellulose membranes and analyzed by immunoblotting with anti-ND2,anti-Src or anti-Fyn (mouse monoclonal clone 25, Pharmingen,Mississauga, Ontario). Bound antibody was then detected on film usingappropriate secondary antibody/HRP conjugates and an ECL kit (AmershamPharmacia Biotech). For control immunoprecipitations under denaturingconditions, SDS was added to the initial protein samples to a finalconcentration of 0.4% and the samples were boiled for 5 minutes andrapidly cooled to 4° C. prior to the addition of the antibodies used forimmunoprecipitation. In addition, pre-adsorption of the anti-ND2antibody with antigenic peptide prevented antibody signal detection onimmunoblots.

Non-receptor tyrosine kinase Src and ND2 are both expressed in cells ofmultiple, diverse tissues. Illustrative, albeit non-limiting, examplesare peripheral nervous system tissue, central nervous system tissue,heart, intestine, kidney, liver, lung, pancreas, skeletal muscle,spleen, testis, bone, skin and brain. The data presented in FIGS. 10A-Bshows that ND2 and Src interact in multiple, diverse tissues.Immunoblots of co-immunoprecipitates from various tissues (FIG. 10A) andtissue homogenates (FIG. 10B) probed with anti-ND2, anti-Src, oranti-Fyn as indicated. Tissues: B—brain; H—heart; I—intestine; K—kidney;Liv—liver; Lu—lung; P—pancreas; Sk—skeletal muscle; Sp—spleen andT—testis. Non-specific IgG applied to liver homogenate was used as anegative control for co-immunoprecipitation. Immunoblotting of Fynprotein from brain was used as a positive control for the anti-Fynantibody. In these experiments the cell lysates were prepared usingnon-denaturing conditions, but when denaturing conditions were used toprepare the proteins, no co-immunoprecipitation of Src by anti-ND2 or ofanti-Src was found (data not illustrated).

Example 9

Src40-49Tat (TSUDAPI-1) inhibits neuropathic pain

Increased activity of NMDA receptors is known to play a major role inpain produced by peripheral nerve injury (Ren et al. Journal ofOrofacial Pain 13:155-163 1999). This type of pain is debilitating andtreatments remain relatively ineffective. Antagonists of the NMDAreceptor complex have been suggested as potential drugs for neuropathicpain management (Planells-Cases et al. Mini Review of MedicinalChemistry 3(7):749-756 2003). However, non-selective blocking of NMDAreceptor function is deleterious, since complete blockade of synaptictransmission mediated by NMDA receptors is known to hinder neuronalsurvival (Ikonomidou et al. Lancet: Neurology 1:383-386 2002; Fix et al.Experimental Neurology 123:204 1993; Davis et al. Stroke 31:347 2000;Morris et al. Journal of Neurosurgery 91:737 1999). The method of theinstant invention selectively blocks NMDAR-mediated excitatorypost-synaptic current (EPSC) without effecting the AMPA(GluR1-containing α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid)component.

Pain induced by cuff implantation, in a laboratory animal such as a rator mouse, is an art-accepted model of neuropathic pain. Generally, incuff implantation a nerve root in the sciatic nerve leading to a hindpaw is tied off by surgical implantation of a “cuff”, for example, apolyethylene ring. Over a period of time the nerve degenerates and aneuropathic pain pattern develops. A control is created by subjectinganother group of animals to “sham surgery”, a procedure wherein theanimals receive the same type of surgery as cuff implantation withoutthe physical implantation of the cuff. After a period of time, the pawis stimulated by a series of filaments or exposure to a small amount ofmoderate heat. Pain is measured by observation of “paw withdrawal”, ingeneral, if the animal lifts the paw and the time it takes to do so.Typically, an animal not experiencing neuropathic pain will not respondto the stimuli. When testing using filaments, threshold is defined interms of force. A reduction in threshold suggests the development ofallodynia. This testing method is described in the art; RenPhysiological Behavior 67:711-716 1999 and Guo et al. The Journal ofNeuroscience 22(14):6208-6217 2002.

In this experiment, tactile allodynia was induced in a group of mice bycuff implantation and symptoms were observed as early as day 3post-surgery. Allodynia was allowed to fully develop at 8-10 dayspost-surgery before the “paw withdrawal” tests were performed. A groupof mice received an intrathecal (introduced into the space under thearachnoid membrane of the brain or spinal cord) injection of 0.02 pmolof Src40-49Tat (TSUDAPI-1, SEQ ID NO:2), a second group of mice receivedan intrathecal injection of 0.02 pmol Scramble Src40-49Tat and a thirdgroup of mice received an intrathecal injection of saline. “ScrambleSrc40-49Tat” refers to TSUDAI-1 (SEQ ID NO:2) having a “scrambled”sequence, i.e. having amino acid residues out of order from the normal.The results are presented in FIG. 11. Green represents Src40-49Tat(TSUDAPI-1, SEQ ID NO:2), grey represents Scramble Src40-49Tat and redrepresents saline. Src40-49Tat (TSUDAPI-1, SEQ ID NO:2), but notScramble Src40-49Tat or saline, significantly reversed allodynia. Thereversal effect was observed as early as one hour followingadministration. The reversal was significant at more than 5 hours postinjection, with PWT 6.03±1.45 g (Src40-49Tat) versus 1.7±0.47 g(scrambled Src40-49Tat)(p<0.05).

Another group of mice received an intravenous injection of 10 pmol/g ofSrc40-49Tat (TSUDAPI-1, SEQ ID NO:2) and a second group of mice receivedan intravenous injection of saline. The results are presented in FIG.12. Green represents Src40-49Tat (TSUDAPI-1, SEQ ID NO:2) and redrepresents saline. Src40-49Tat (TSUDAPI-1, SEQ ID NO:2), but not saline,significantly reversed allodynia. The reversal effect was observed asearly as one hour following administration. The anti-allodynic effect ofthe peptide (SEQ ID NO:2) peaked at 2 hours following injection, withPWT 9.28±2.55 g (Src40-49Tat) versus 1.95±0.617 g (saline) (p<0.05).

Protein phosphorylation is a major mechanism for receptor function. Itis known that tyrosine phosphorylation of the NR2 subunits plays a keyrole in NMDA receptor activation (Moon et al. PNAS USA 91:3954-39581994; Lau et al. Journal of Biological Chemistry 270:20036-20041 1995;Xiong et al. Journal of Neuroscience 19:RC37(1-6) 1999). It is alsoknown that inflammatory hyperalgesia is associated with rapid andprolonged enhancement of tyrosine phosphorylation of the NR2B subunitsof NMDA receptors (Guo et al. The Journal of Neuroscience22(14):6208-6217 2002).

In this experiment, a significant increase of tyrosine phosphorylationof NMDA receptor NR2B subunits in spinal cord dorsal horn tissue wasobserved in cuffed rats but not in sham-operated rats (FIG. 15A).Intrathecal injection of 0.02 pmol Src40-49Tat (TSUDAPI-1, SEQ ID NO:2)significantly reversed the increase in tyrosine phosphorylation (FIG.15B). FIG. 15A shows a western blot of an immunoprecipitation using ananti-NR2B antibody and anti-phosphorylated tyrosine antibody. FIG. 15Bshows a graph quantifying the tyrosine phosphorylation calculated as apercent of the control. This graph represents quantification of band(from western blots) density in three experiments.

Another experiment was performed to compare cuff-induced allodynia inwild-type mice to Src kinase null mice (Src kinase knock-out mice). Theresults are shown in FIG. 13. The PWT was tested in the mice prior tosurgery. The basal PWT in Src kinase null mice was not different fromthat of wild-type mice; see black bars in graphs in center and righthand side in FIG. 13, Src−/− and Src+/+. Both groups of mice received anintravenous injection of 100 pmol of Src40-49Tat (TSUDAPI-1, SEQ IDNO:2). Allodynia was depressed in Src kinase null mice throughout thetesting time course, with PWT 0.01±1.53 g (wild) versus 0.21±0.04 g(null) (p<0.05) at 22 days post surgery (left graph in FIG. 13).Treatment with Src40-49Tat (100 pmol, intravenous injection)significantly reversed allodynia in wild-type mice from 0.008±0.0 g to0.23±0.05 g (p<0.05), but did not further increase PWT in null mice.Nerve-injury induced increase in phosphorylation of the NR2B subunit wasfound to be depressed in both wild-type and Src kinase null mice.Treatment with Src40-49Tat did not further decrease phosphorylation ofNR2B subunits in Src kinase null mice.

Thus, the instant inventors concluded that loss of Src or depressedaction of Src through treatment with Src40-49Tat (TSUDAPI-1, SEQ IDNO:2) inhibits Src-mediated NMDAR up-regulation dependent neuropathicpain.

IN SUMMARY

The main criteria for identifying ND2 as the protein mediating theinteraction between NMDARs and the unique domain of Src, as inferredfrom previous work (Ali et al. Current Opinion in Neurobiology11:336-342 2001; Yu et al. Science 275:674-678 1997) are as follows: ND2must bind directly to the unique domain of Src through amino acids 40-58(specifically 40-49; SEQ ID NO:1); this binding must be prevented by theSrc40-58 (specifically 40-49) peptide; ND2 must be present at excitatorysynapses and must be a component of the NMDAR complex; and lack of ND2must prevent the upregulation of NMDAR activity by endogenous Src.

ND2 was first considered as a potential Src unique domain-bindingprotein when overlapping clones of ND2 in two separate yeast two-hybridexperiments were isolated. Subsequently, the direct interaction of theSrc unique domain and ND2 was confirmed through in vitro binding assaysusing recombinant proteins. Through these experiments the ND2.1 regionwas identified as necessary and sufficient for interacting with the Srcunique domain. ND2.1 bound directly to the Src40-58 (specifically 40-49)peptide and the in vitro binding of the Src unique domain to ND2.1 wasprevented by Src40-58 (specifically 40-49). Src and ND2co-immunoprecipitated with each other in brain homogenates and PSDprotein preparations. The co-immunoprecipitation was prevented bySrc40-58 (specifically 40-49), implying that the Src-ND2 interactionidentified in vitro may occur in vivo. In addition to finding ND2 in PSDprotein preparations, ND2-immunoreactivity was found by immunogoldelectron microscopy in PSDs in the CA1 hippocampus. Moreover,co-immunoprecipitation experiments indicated that ND2 is a component ofthe NMDAR complex and that the Src-ND2 interaction is required for theassociation of Src, but not ND2, with NMDARs. It was found thatdepleting ND2 suppresses Src association with the NMDAR complex andprevents the upregulation of NMDAR function by activating endogenous Srcat excitatory synapses. Src40-49 (SUDAPI-1; SEQ ID NO:1) was identifiedas the specific peptide that interacts with ND2 as Src50-58 alone didnot interact with ND2. Finally, it was found that TAT-Src40-49(TSUDAPI-1; SEQ ID NO:2) as administered to rats reduced pain behaviorin the formalin test. These multiple, converging lines of evidence leadto the conclusion that ND2 is the protein mediating the interactionbetween NMDARs and the unique domain of Src.

ND2 is mitochondrially encoded and translated, and yet it is foundwithin the PSDs of glutamatergic synapses in the brain. The othermitochondrial proteins examined, ND4 and Cyto 1, were not detected inthe PSD fraction implying that this fraction is not contaminatednon-specifically by mitochondrial proteins. Further,ND2-immunoreactivity by immunogold electron microscopy was found withinstructurally-identified PSDs in dendritic spines of CA1 neurons. In thispreparation, proteins are immobilized by tissue fixation precluding thepossibility that ND2 could have relocated from the mitochondria to thePSD during processing. Moreover, because dendritic spines are devoid ofmitochondria (Shepherd et al. Journal of Neuroscience 18(20):8300-83101998) the ND2 immunoreactivity cannot be accounted for by mitochondriaabutting the PSD. Taken together these findings indicate that ND2, butnot the entire Complex I, is normally present within the PSD. The PSDcontains many enzymes that may be involved in regulating synapticfunctioning (P. Siekevitz Proceedings of the National Academy of ScienceUSA 82:3494-3498 1985) including glycolytic enzymes capable ofgenerating ATP (Wu et al. Proceedings of the National Academy of ScienceUSA 94:13273-13278 1997). However, without other components of Complex Iit is unlikely that ND2 functions catalytically within the PSD.

Thus, in addition to its localization in mitochondria and function as acomponent of Complex I, the present results indicate that ND2 has asecond location and function in outside the mitochondria. Mitochondriaare intimately linked to overall cellular functioning through generationof ATP by oxidative phosphorylation. Mitochondria are also known to bekey for sequestration of intracellular calcium (D. D. Friel Cell Calcium28:307-316 2000; R. Rizzuto Current Opinion in Neurobiology 11:306-311)and to participate in programmed cell death (Gorman et al. DevelopmentalNeuroscience 22:348-358 2000; M. P. Mattson National Review of Molecularand Cellular Biology 1:120-129 2000). Some mitochondrial proteins areknown to be present at extra-mitochondrial sites (Soltys et al. Trendsin Biochemical Science 24:174-177 1999; Soltys et al. InternationalReview of Cytology 194:133-196 1999). But, the experiments describedherein indicate a new type of function for a mitochondrial proteinoutside this organelle, that is ND2 acts as an adapter protein thatanchors Src within the NMDAR complex, where it thereby allows Src toupregulate NMDAR activity.

Upregulating the activity of NMDARs is a major function of Src inneurons in the adult CNS (Lu et al. Science 279:1363-1368 1998; Pelkeyet al. Neuron 34:127-138 2002; Huang et al. Neuron 29:485-496 2001) andthis mediates the induction of long-term potentiation (LTP) ofexcitatory synaptic transmission in CA1 neurons in the hippocampus (Aliet al. Current Opinion in Neurobiology 11:336-342 2001). The findingsdescribed herein imply that the ND2-Src interaction is essential for LTPinduction as LTP in CA1 neurons is prevented by Src40-58 and byanti-Src1, an antibody that recognizes this amino acid sequence withinthe Src unique domain and which prevents the Src unique domaininteraction with ND2.1 in vitro (J. R. G., M. W. S. unpublishedobservations). LTP at Schaffer collateral-CA1 synapses is the prototypicexample of NMDAR-dependent enhancement of excitatory synaptictransmission, which is observed at numerous types of glutamatergicsynapses throughout the CNS (Malenka et al. Science 285:1870-1874 1999).In addition, Src has been implicated in NMDAR-dependent seizures (Sannaet al. Proceedings of the National Academy of Science 97:8653-86572000), chronic pain (Guo et al. Journal of Neuroscience 22:6208-62172002) and neurotoxicity (Pei et al. Journal of Cerebral Blood FlowMetabolism 21:955-963 2001). Thus, the discovery of the Src-ND2interaction at NMDARs, which is disclosed herein, defines aprotein-protein interaction of general relevance to regulation ofneuronal function, synaptic plasticity, and pathophysiology in the CNS.

Additionally, by showing an extramitochondrial action for a proteinencoded in the mitochondrial genome a previously unsuspected means bywhich mitochondria regulate cellular function has been identified.Because ND2 and Src are broadly expressed, the interaction of ND2 withthe Src unique domain may be of general relevance for control of Srcsignaling (Example 8 and FIGS. 10A-B).

All patents and publications mentioned in this specification areindicative of the levels of those skilled in the art to which theinvention pertains. All patents and publications are herein incorporatedby reference to the same extent as if each individual publication wasspecifically and individually indicated to be incorporated by reference.

It is to be understood that while a certain form of the invention isillustrated, it is not to be limited to the specific form or arrangementherein described and shown. It will be apparent to those skilled in theart that various changes may be made without departing from the scope ofthe invention and the invention is not to be considered limited to whatis shown and described in the specification. One skilled in the art willreadily appreciate that the present invention is well adapted to carryout the objectives and obtain the ends and advantages mentioned, as wellas those inherent therein. The oligonucleotides, peptides, polypeptides,biologically related compounds, methods, procedures and techniquesdescribed herein are presently representative of the preferredembodiments, are intended to be exemplary and are not intended aslimitations on the scope. Changes therein and other uses will occur tothose skilled in the art which are encompassed within the spirit of theinvention and are defined by the scope of the appended claims. Althoughthe invention has been described in connection with specific preferredembodiments, it should be understood that the invention as claimedshould not be unduly limited to such specific embodiments. Indeed,various modifications of the described modes for carrying out theinvention which are obvious to those skilled in the art are intended tobe within the scope of the following claims.

1. A method for ameliorating pain in a subject by modifyingN-methyl-D-aspartate receptor (NMDAR) interaction with non-receptortyrosine kinase Src in cells of said subject comprising: administering acomposition including TSUDAPI-1 (SEQ ID NO:2) to said subject in anamount effective to achieve modification of said NMDAR interaction withnon-receptor tyrosine kinase Src in said cells wherein said modificationameliorates pain in said subject.
 2. The method as in claim 1 whereinsaid pain is inflammatory pain.
 3. The method as in claim 1 wherein saidpain is neuropathic pain.
 4. A method for ameliorating pain in a subjectcomprising administering a peptide fragment of the Src unique domain,wherein the Src unique domain occupies residues 4-82 of SEQ ID NO:4, andthe peptide fragment inhibits an interaction occurring between the Srcunique domain and ND2, and the peptide fragment is attached to a carriereffective to transport the peptide fragment into cells to the subject,whereby pain is ameliorated in the subject.
 5. The method of claim 4,wherein the amino acid sequence of the peptide fragment consists of SEQID NO:1.
 6. The method of claim 4, wherein the amino acid sequence ofthe peptide fragment attached to the carrier consists of SEQ ID NO:2. 7.The method of claim 4, wherein the pain is inflammatory pain.
 8. Themethod of claim 4, wherein the pain is neuropathic pain.